Journal: Journal of Biological Chemistry
Article Title: Protection of telomeres 1 proteins POT1a and POT1b can repress ATR signaling by RPA exclusion, but binding to CST limits ATR repression by POT1b
doi: 10.1074/jbc.ra118.004598
Figure Lengend Snippet: Figure 3. ATR repression by POT1b is limited by its interaction with CST. A, schematic of the role of POT1b in recruiting CST and polymerase /primase to telomeres (top). Schematic of POT1b domains and the mutations that disrupt the POT1b-CST interaction (bottom). B, immunoblot for POT1b in POT1aF/F MEFs expressing the indicated proteins before and after treatment with Cre (96 h). ctrl, nonspecific band used as loading control. C, representative IF-FISH images showing the DNA damage response at telomeres in Cre-treated POT1aF/F MEFs expressing POT1a, POT1b, or POT1bCST (96 h). H2AX foci that co-localize with telomeres (TIFs) were detected using -H2AX antibody (red) and a telomeric FISH probe (green). D, quantification of the TIF response as shown in C of POT1aF/F MEFs before and after Cre treatment. Bar graphs represent averages from three experiments and S.D.s. p values were derived from a two-tailed Student’s t test. p values: **, p 0.01; *, p 0.05. E, immunoblot for POT1a, POT1b, and Stn1 in POT1aF/F MEFs (with and without 96 h Cre treatment) expressing exogenous POT1b and/or Stn1 shRNA (indicated with ) or a scrambled sh (indicated with ). Asterisks indicate nonspecific bands. ctrl, nonspecific band used as loading control. F, representative images of IF-FISH to monitor the DNA damage response at telomeres in the indicated Cre-treated (96 h) POT1aF/F MEFs with and without POT1b and expressing Stn1sh or a scrambled control (ctrl sh). H2AX foci that co-localize with telomeres (TIFs) were detected with IF with an -H2AX antibody (red) and FISH with a telomeric PNA probe (green). G, quantification of TIF response in the indicated POT1aF/F MEFs (as in F). Bar graphs represent averages from three independent experiments and S.D.s. p values: **, p 0.001; *, p 0.05; ns, not significant.
Article Snippet: After immunoblotting, the membranes were blocked in TBS with Tween 20 with 2.5% nonfat dry milk and incubated with the following primary antibodies -POT1a (no. 1221), -Myc (9B11, Cell Signaling Technology), -TIN2 (no. 1447), -Rap1 (no. 1253), -RPA32 (A300 –244A, Bethyl), -RPA70 (A300 –241A, Bethyl), -POT1b (no. 1223), -STN1 (sc376450, Santa Cruz Biotechnology), -tubulin (GTU88, Abcam) in 1% nonfat dry milk.
Techniques: Western Blot, Expressing, Control, Derivative Assay, Two Tailed Test, shRNA